Methods for treating ocular diseases via introxide and or polyhydroxy acid containing compounds

ABSTRACT

Methods for prevention or treatment of ocular diseases via administration of a nitroxide and/or polyhydroxy acid containing compound are provided.

FIELD OF THE INVENTION

The present invention relates to new uses for compositions comprising anitroxide and/or polyhydroxy acid containing compounds for treatment ofocular disease such as macular degeneration, cataracts and glaucoma.

BACKGROUND OF THE INVENTION

Nitroxides are stable free radicals with antioxidant catalyticactivities similar to superoxide dismutase. Nitroxides existing in vivohave been shown to interact with other substances to also mimic catalaseactivities. Thus, nitroxide containing compounds have been described inthe art for numerous uses. For example, U.S. Pat. No. 5,462,946discloses biologically compatible compositions containing an effectiveamount of a metal independent nitroxide compound for use in protectingthe skin against ionizing radiation, mucositis, the effects of wholebody radiation and radiation induced hair loss. In this embodiment, thenitroxide containing composition is applied topically as an ointment,lotion or cream, intravenously or orally by pill or lozenge. This patentalso teaches the nitroxide containing compounds to be useful asprotectants against: increased oxygen exposure so as to avoid pulmonaryadult respiratory distress syndrome; oxygen-induced lenticulardegeneration and hyaline membrane disease in infants; oxidativestress-induced cataracts; reperfusion injury in treating cardiovascularphenomena such as myocardial infarction and strokes, pancreatitis orintestinal ulceration and organ transplant; cytotoxicity due to excessoxidation in animal or plant cell cultures; cytotoxic effects ofchemotherapeutic agents; and mutagenic and carcinogenic agents. Alsotaught in this patent is use of these compounds as anti-inflammatoryagents effective against arthritic conditions. In this embodiment, thenitroxide containing compositions are administered parenterally,intra-articularly or via oral ingestion. This patent also teaches use ofthese compounds as aging retardants when administered orally orparenterally and in weight reduction when administered orally orintravenously.

U.S. Pat. Nos. 5,824,781, 5,840,701 and 5,817,632 teach compositions andprocesses to alleviate free radical toxicity based on use of nitroxidesin association with physiologically compatible macromolecules. Thesecompositions are suggested to be useful as blood substitutes,radioprotective agents, imaging agents, agents to protect againstischemia and reperfusion injury, particularly cerebral stroke, and invivo enzyme mimics.

Benzo-fused heterocyclic polyhydroxy acid containing compounds for usein treating dermatological, rheumatic, respiratory, cardiovascular andcorneopathies are taught in U.S. Pat. No. 5,849,798 and U.S. Pat. No.6,194,450. Phenyl benzoate derivatives with polyhydroxy acidsubstitutions for use in medicines and cosmetics for treatingdermatological conditions relating to keratinization disorders, asantiinflammatories and as anti-immuno-allergic agents are taught in U.S.Pat. No. 5,332,856 and U.S. Pat. No. 5,468,897.

Methods and compositions containing hydroxyacids or related compoundsfor enhancing the therapeutic effects of cosmetic and pharmaceuticalagents used in topical treatment of cosmetic conditions, dermatologicaldisorders and other afflictions are described in U.S. Pat. No. 6,051,609and U.S. Pat. No. 5,962,526.

SUMMARY OF THE INVENTION

An object of the present invention is to provide pharmaceuticalcompositions for prevention and/or treatment of ocular diseases such asmacular degeneration, cataracts and glaucoma comprising a nitroxideand/or polyhydroxy acid containing compound and a pharmaceuticallyacceptable vehicle.

Another object of the present invention is to provide methods forprevention and/or treatment of ocular diseases such as maculardegeneration, cataracts, and glaucoma via administration of a nitroxideand/or polyhydroxy acid containing compound.

DETAILED DESCRIPTION OF THE INVENTION

Macular degeneration is a disease affecting the macula, a thin area inthe center of the retina that confers the ability for acute and detailedvision. The central area of the macula is known as the fovea and iscomposed entirely of cones. These cones have specialized structure thataids in detection of detail in a visual image. In this region, the bloodvessels, ganglion cells, and plexiform layers are all displaced to oneside to allow light to pass unimpeded to the cones.

Degeneration of the tissue in this area of the retina is primary causeof debilitating visual impairment in the elderly and a major cause ofblindness in developing countries throughout the world. Two forms of thedisease have been identified, geographic atrophy and choroidalneovascularization. This disease is most often linked to aging. However,macular degeneration can also result from hypertension in youngerindividuals.

Treatments currently used in patients with the choroidalneovascularization form of macular degeneration include photodynamictherapy, pharmacologic inhibition of choroidal neovascular membraneformation with anti-angiogenic agents, surgical intervention, includingexcision of subfoveal choroidal neovascular membranes, and radiationtherapy (Ciulla et al. Surv. Opthalmol. 1998 43:134-146). The goal oftherapy in each method of treatment is to destroy the choroidalneovascular membranes.

A cataract is a lens opacity and may be congenital, traumatic, orsecondary to a systemic disease such as diabetes, myotonic dystrophy, oratopic dermatitis, systemic corticosteroid treatment, or uveitis. Senilecataract is the most common type withe most persons over the age of 60having some degree of lens opacity. Primary treatment is surgicalremoval of the cataract.

Glaucoma is a disease of the eye characterized by increased intraocularpressure due to restricted outflow of the aqueous humor through theaqueous veins and Schlemm's canal, excavation and degeneration of theoptic disk, and nerve fiber bundle damage producing arcuate defects inthe field of vision. Treatments include topical administration ofpharmacological agents such as β-adrenergic blocking agents such astimolol, levobunolol and metipranolol, β₁-receptor selective blockingagents such as betaxolol, epinephrine, α₂-agonists such as apraclonide,pilocarpine, prostaglandin analogues such as latanoprost, carbonicanhydrase inhibitors such as dorzolamine and acetazolamide. Additionaltreatments include laser trabeculoplasty as well as surgicaltrabeculectomy.

It is now believed that application of a composition comprising anitroxide and/or polyhydroxy acid containing compound will be useful intreating ocular diseases such as macular degeneration, cataracts, andglaucoma. For purposes of the present invention, by “nitroxide” or“nitroxide containing compound” it is meant stable nitroxide freeradicals. Examples of nitroxide containing compounds are well known inthe art and taught in prior art references such as U.S. Pat. No.5,462,946.

For purposes of the present invention, by “polyhydroxy acid” or“polyhydroxy acid containing compound” it is meant a compound,preferably an aromatic compound, with a polyhydroxy acid substitution.Exemplary polyhydroxy acid containing compounds are well known in theart and taught in prior art references such as U.S. Pat. No. 6,051,609and U.S. Pat. No. 5,962,526.

Phototoxicity and cytotoxicity as well as the the ability of nitroxideand/or polyhydroxy acid containing compounds to protect againultraviolet-induced gene induction were examined in human skinfibroblasts containing the human elastin promoter linked to a luciferasereporter gene construct. Glucolactone did not demonstrate significantphototoxicity or cytotoxicity, with the ED₅₀ for phototoxicity beingapproximately 102 g/L. Further glucolactone protected against UV-inducedpromoter induction approximately 50%. Thus, gluconolactone protectsagainst UV-induced gene induction in cells containing the human elastinpromoter/luciferase construct. Tempol also demonstrated protectionagainst increasing amounts of ultraviolet radiation in the neutral redassay. The ED₅₀ for cell cultures with tempol but not receiving anyultraviolet radiaiton was approximately 100 mM. The ED₅₀ for cellcultures with tempol and receiving ultraviolet radiation doses of 2 and5 mJ/cm² was approximately 150 and 250 mM, respectively. Further, atincreasing ultraviolet doses tempol protected significantly againsttoxicity in both the neutral red assay and the human elastinpromoter/luciferase assays, demonstrating over 50% protection.

Accordingly, these experiments are indicative of nitroxide and/orpolyhydroxy acid containing compounds providing a safe means forprotecting against toxicities of ultraviolet radiation in cells andproviding useful treatment for oculat disease such as maculardegeneration, cataracts and/or glaucoma.

Nitroxide and/or polyhydroxy acid containing compounds can be formulatedwith a pharmaceutically acceptable vehicle selected in accordance withthe route of administration. For example, for production of apharmaceutical composition which can be applied directly to the eye, thecompositions may be formulated either as a cream or in solution; fororal administration, the compositions may be formulated as a tablet,capsule or in solution; for intravenous administratation oradminstration via injection parenterally or intramsucularly, thecompound is prefereably dissolved in saline or phosphate bufferedsaline, etc. Formulations of such pharmaceutical compositions can beproduced routinely by one of skill in the art in accordance with wellknown techniques. In one embodiment of the present invention, thepharmaceutical compositions comprises either a nitroxide containingcompound or a polyhydroxy acid containing compound. More preferred arepharmaceutical compositions comprising both a nitroxide containingcompound and a polyhydroxy acid containing compound.

These pharmaceutical compositions can be administered to a patientsuffering from an ocular disease such as macular degeneration,cataracts, and glaucoma for treatment thereof. These pharmaceuticalcompositions can also be adminsitered to a patient to prevent or slowdevelopment of ocular diseases such as macular degeneration, cataracts,and glaucoma. In a preferred embodiment, the compositions isadministered directly to the eye. However, the composition can also beadministered orally, intravenously, sublingually, intramuscularly, viasuppository, or any other route wherein the compounds maintain theirability to treat the ocular disease. For purposes of the presentinvention, by treatment, treat to treating, it is meant an alleviationor decrease in the symptoms of a patient suffering from maculardegeneration.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Fibroblast Cultures

Immortalized human skin fibroblasts containing the human elastinpromoter linked to a luciferase reporter gene construct were seeded in96-well plates at a density of 7,500 cells per well (doubling time ˜30hours). Cells were grown in 75 cm² tissue culture flasks in MEM (withoutphenol red) supplemented with 10% FBS, 25 mM HEPES, 2 mM l-glutamine, 1%nonessential amino acids, 50 μg/mL Gentamycin, and 5 μg/mL Zeocin. Cellcultures were incubated in a 37° C. incubator (containing 5% CO₂) whennot being manipulated or irradiated. Cell cultures were washed withPhosphate Buffered Saline (PBS) with Ca⁺⁺ and Mg⁺⁺ when media orcompounds were removed and before ultraviolet radiation (UVR).

Example 2 Gluconolactone and Tempol

Gluconolactone was prepared in PBS. Concentrations of 0.5, 1.0, 2.5,5.0, 7.5, 10.0, 12.5, and 25% (w/v) were prepared and adjusted to a pHof 7.2. Gluconolactone was incubated 15 minutes prior to and in contactwith cell cultures during UV-irradiation.

4-Hydroxy-tempo (tempol) was prepared in PBS. Concentrations of tempolof 10, 25, 50, 62.5, 100, 125, 250, and 500 mM were prepared andadjusted to a pH of 7.2. Tempol was incubated 15 minutes prior to and incontact with cell cultures during UV-irradiation.

Example 3 Ultraviolet Radiation

Cell cultures were UV-irradiated with Westinghouse 40-Watt fluorescentsunlamps (FS-40 lamps) with doses of either 2 or 5 mJ/cm², based on aPMA2100 radiometer (Solar light Company, Philadelphia, Pa.) equippedwith a PMA2101 erythema weighted detector.

Example 4 Neutral Red Cytotoxicity/Phototoxicity Assay

Fifteen hours after UV-irradiation with the FS-40 lamps, cell cultureswere washed with PBS and Neutral Red Medium (Neutral Reddye+supplemented MEM) was added for a 3 hours (in 37° C. incubators).After the incubation period, cells are washed with PBS and the NeutralRed Desorb solution (50% Ethanol, 49% distilled water, 1% Glacial AceticAcid) was added. Plates are shaken for 10 minutes at 500 rpm and thenthe absorption was read at 540 nm using THERMOmax plate reader andSOFTmax PRO software.

1. A method for treatment of ocular diseases comprising administering toa patient suffering from an ocular disease a nitroxide containingcompounds or polyhydroxy acid containing compounds.
 2. The method ofclaim 1 wherein the ocular disease is macular degeneration, cataracts,or glaucoma.
 3. A method for treatment of ocular diseases comprisingadministering to a patient suffering from an ocular disease a nitroxidecontaining compound and a polyhydroxy acid containing compound.
 4. Themethod of claim 3 wherein the ocular disease is macular degeneration,cataracts, or glaucoma.
 5. A method for preventing ocular diseasescomprising administering to a subject a nitroxide containing compoundsor polyhydroxy acid containing compounds.
 6. The method of claim 5wherein the ocular disease is macular degeneration, cataracts, orglaucoma.
 7. A method for preventing ocular diseases comprisingadministering to a subject a nitroxide containing compound and apolyhydroxy acid containing compound.
 8. The method of claim 7 whereinthe ocular disease is macular degeneration, cataracts, or glaucoma.
 9. Apharmaceutical composition for treatment or prevention of oculardiseases comprising a nitroxide containing compound, a polyhydroxy acidcontaining compound, and a pharmaceutically acceptable vehicle.
 10. Thepharmaceutical composition of claim 9 wherein the ocular disease ismacular degeneration, cataracts, or glaucoma.